Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

A ring trial to harmonize Toxoplasma gondii microsatellite typing: comparative analysis of results and recommendations for optimization.

A ring trial among five European laboratories was organized to reach consistency in microsatellite (MS) typing of the zoonotic parasite Toxoplasma gondii. Three sample sets were circulated and analyzed by each laboratory following a previously published method that is based on fragment length polymorphism of 15 MS markers. The first sample set compared typing results in general and focused on effects of DNA concentration; the second sample set focused on the polymorphic fingerprinting markers that can differentiate T. gondii strains within the same archetypal lineage; and the third set focused on non-archetypal genotypes. Methodological variations between laboratories, including the software programs used to determine MS fragment length, were collated using a questionnaire. Overall, lineage-level typing results reached a high level of agreement, especially in samples with the highest DNA concentrations. However, laboratory-specific differences were observed for particular markers. Major median differences in fragment length, of up to 6 base pairs, were related to the fluorophore used to label fragment-specific primers. In addition, primer pairs with identical sequences obtained from different suppliers resulted in fragments of differing length. Furthermore, differences in the way the sequencing profiles were assessed and interpreted may have led to deviating results in fragment length determination. Harmonization of MS typing, for example, by using the same fluorophores or by numerical adjustments applied to the fragment-lengths determined, could improve the uniformity of the results across laboratories. This is the first interlaboratory comparison, providing guidelines (added as a supplement) for the optimization of this technique.

Spinal cord toxoplasmosis in a young immunocompetent patient.

We report a case of spinal cord toxoplasmosis occurring as a primary infection in a 31-year-old immunocompetent man. Exhaustive immunologic and genetic investigations did not identify any immunodeficiency. The causative agent was a typical type 2 strain. In cases of spinal cord lesions, toxoplasmosis should be considered, even in an immunocompetent patient.

[Seroprevalence of Toxoplasmosis among Pregnant Women in Benin: Meta-Analysis and Meta-Regression]. / Séroprévalence de la toxoplasmose chez les femmes enceintes au Bénin : méta-analyse et métarégression.

To assess the seroprevalence of toxoplasmosis among pregnant women in Benin, we conducted a meta-analysis using the PRISMA criteria. Al research published between 1990 and 2018 on toxoplasmosis among pregnant women Benin were eligible. A total of five databases were investigated, and the extracted data were subjected to a meta-analysis under R 3.1 using both random effect model and fixed effect model. The overall prevalence of toxoplasma-specific IgG among pregnant women was 47% (CI 95%: 40-53) and that of specific IgM was 2% (CI 95%: 1-3). The infection rate in urban areas (52%) was significantly higher than in rural areas (33%). The two main risk factors identified by the various eligible studies were the age of the pregnant women and the consumption of raw vegetables. We show that toxoplasmosis is endemic in pregnant women in Benin, implying that primary prevention measures must be put in place by the competent authorities to control this infection.

Afin d'évaluer le niveau de l'infection toxoplasmique chez les femmes enceintes au Bénin, nous avons effectué une méta-analyse selon le protocole PRISMA. Étaient éligibles tous les articles de recherche publiés entre 1990 et 2018 sur la toxoplasmose chez les femmes enceintes en consultation prénatale au Bénin. Au total, cinq bases de données ont été consultées, puis les données extraites ont été soumises à une méta-analyse sous R 3.1 selon les modèles à effet aléatoire et à effet fixe. La séroprévalence de la toxoplasmose chez la femme enceinte était de 47 % (IC 95 % : 40­53) pour les IgG et de 2 % (IC 95 % : 1­3) pour les IgM spécifiques. Le taux d'infection en milieu urbain (52 %) était significativement plus élevé qu'en milieu rural (33 %). Deux principaux facteurs de risque associés à la toxoplasmose ont été identifiés par les différentes études éligibles : l'âge des gestantes et la consommation de crudités. Nous montrons ainsi que la toxoplasmose est endémique chez les femmes enceintes au Bénin, impliquant que des mesures de prévention primaire soient mises en place par les autorités compétentes pour contrôler cette infection.

Diversity of Toxoplasma gondii strains at the global level and its determinants.

The population structure of Toxoplasma gondii is characterized by contrasting geographic patterns of strain diversity at different spatial scales: global, regional and even local scales in some regions. The determinants of this diversity pattern and its possible evolutionary mechanisms are still largely unexplored. This review will focus on three main dichotomies observed in the population structure of the parasite: (1) domestic versus wild, (2) South America versus the rest of the world and (3) intercontinental clonal lineages versus regional or local clonal lineages. Here, the impact in terms of public health of this remarkably contrasting geographic diversity of T. gondii populations is discussed, with emphasis on the role of globalization of exchanges that could lead to rapid evolution of T. gondii population spatial structure and new challenges in a One Health context.

Geographical distribution of Toxoplasma gondii genotypes in Asia: A link with neighboring continents.

Defining the pattern of genetic diversity of Toxoplasma gondii is important to understand its worldwide distribution. During the last decades, a large number of studies have been published on Toxoplasma genotypes circulating in Europe, in North and South America. Two continents are still largely unexplored, Africa and, to a less extent, Asia. In this last continent, an increasing number of publications reported genotypes circulating in diverse provinces of China, but very few data are available for other Asian countries. After a systematic database search, 47 papers related to T. gondii genotypes in Asia were analyzed. Genetic characterization of DNA was performed by microsatellite markers, or more usually by a multiplex PCR using 11 PCR-RFLP markers, allowing data comparison to draw a first global picture of the population structure of this parasite throughout Asia. Overall, 390 isolates or DNA extracts were completely typed by PCR-RFLP and/or microsatellite marker methods, revealing 36 different PCR-RFLP or equivalent microsatellite genotypes: 15 genotypes identified by a ToxoDB number and 21 atypical or unique genotypes. The most common genotype found in Asia is the genotype ToxoDB#9 (Chinese 1). The clonal types I, II and II variant, and III were also commonly found in Asia. The geographical distribution of these genotypes across Asia may reflect either a continuum with Europe for the western part of Asia (presence of Type II), or the circulation of strains through animal migration or human activities between Africa and the Southwestern part of Asia (Africa 1 genotype in Turkey or ToxoDB#20 both I Sri-Lanka and in Ethiopia or Egypt). Although there are some indications of a genetic population structure in Southeast Asian countries different from the rest of Asia, more studies in this tropical part of Asia will be necessary for a region which represent as well as Africa one of the missing links of the T. gondii genetic diversity.

Epidemiological review of Toxoplasma gondii infection in humans and animals in Portugal.

Toxoplasmosis is a worldwide zoonosis. However, data from Portugal are limited and a considerable part of the literature is in Portuguese. Currently, the rate of congenital infection in Portugal is unknown, and almost nothing is known of sequelae of congenital toxoplasmosis. There is no recent general population-based serological survey of Toxoplasma gondii in humans in Portugal. In addition, there is little information on genetic characteristics of T. gondii in animals and humans. In the present paper, we review prevalence, clinical spectrum and epidemiology of T. gondii in humans and animals in Portugal. This knowledge should be useful to biologists, public health workers, physicians and veterinarians.

Toxoplasma seroconversion with negative or transient immunoglobulin M in pregnant women: myth or reality? A French multicenter retrospective study.

Classically, Toxoplasma infection is associated with high levels of specific IgM antibody and a rise in specific IgG levels 1 to 3 weeks later. Atypical IgG seroconversion, without IgM detection or with transient IgM levels, has been described during serologic follow-up of seronegative pregnant women and raises difficulties in interpreting the results. To evaluate the frequency and the characteristics of these atypical cases of seroconversion, an investigation was conducted within the French National Reference Center for Toxoplasmosis, from which 26 cases collected from 12 laboratories belonging to the network were identified. The aim of this work was to retrospectively analyze the results of serologic testing, the treatments administered, and the results of prenatal and postnatal follow-up for these women. In each case, IgG antibodies were detected using both screening and confirmatory tests. IgM antibodies were not detected in 15 cases, and the levels were equivocal or low-positive in 11 cases. The IgG avidity results were low in 16 cases and high in one case. Most of the pregnant women (22/26) were treated with spiramycin from the time that IgG antibodies appeared until delivery. Amniotic fluid was analyzed for Toxoplasma gondii DNA by PCR in 11/26 cases, and the results were negative in all cases. Congenital toxoplasmosis was ruled out in 12/26 newborns. There was no abnormality observed at birth for 10 newborns and no information available for 4 newborns. In conclusion, when the interpretation of serological results is so difficult, it seems cautious to initiate treatment by spiramycin and to follow the pregnant women and their newborns.

Acute toxoplasmoses in immunocompetent patients hospitalized in an intensive care unit in French Guiana.

Atypical Toxoplasma gondii strains, unrelated to archetypal clonal lineages (I, II, III), have been reported more frequently over the last decade in areas other than Europe and North America. A newly described form of toxoplasmosis, 'Amazonian toxoplasmosis' (AT), has been reported since 2002 in French Guiana. It is characterized by severe cases and atypical strains linked to a neotropical forest-based cycle. We report on the cases of AT that required intensive care management. We performed a prospective observational study on hospitalized adults in the Intensive Care Unit (ICU) from 2002 to 2008. Clinical and laboratory data, microbiological findings and outcomes were recorded. Data, including the ICU simplified acute physiology score and the pneumonia severity index, were calculated. Epidemiological risk factors for AT were assessed through questionnaires. Eleven non-immunodeficient patients were admitted to the ICU in Cayenne for life-threatening pneumonia associated with disseminated toxoplasmosis. Mechanical ventilation was necessary in seven patients, four of whom required immediate orotracheal intubation. Cardiac and ophthalmological abnormalities were found in five and four patients, respectively. One patient died from multiple organ failure. The genetic characterization of Toxoplasma DNA using six microsatellite markers revealed unique and atypical genotypes in eight patients. All patients presented epidemiological risk factors for AT. In French Guiana, significant T. gondii-related infectious syndrome associated with the lungs, a high level of LDH activity and the reported risk factors for AT was strongly suggestive of disseminated toxoplasmosis with a possible trend toward life-threatening pneumonia.

Human impact on genetic diversity of Toxoplasma gondii: example of the anthropized environment from French Guiana.

In French Guiana, severe cases of toxoplasmosis in immunocompetent patients are associated with atypical strains of Toxoplasma gondii linked to a wild neotropical rainforest cycle and a higher genetic diversity than usually observed for T. gondii isolates from anthropized environment. This raises the question of the impact of anthropization of the natural environment, on genetic diversity and on the population structure of T. gondii. However, few data are available on strains circulating in the anthropized areas from French Guiana. Seropositive animals originating mainly from anthropized sub-urban areas and punctually from wild environment in French Guiana were analyzed for T. gondii isolation and genotyping. Thirty-three strains were obtained by bioassay in mice and compared with 18 previously reported isolates chiefly originating from the Amazon rainforest. The genotyping analysis performed with 15 microsatellite markers located on 12 different chromosomes revealed a lower genetic diversity in the anthropized environment. Results were analyzed in terms of population structure by clustering methods, Neighbor-joining trees reconstruction based on genetic distances, F(ST,) Mantel's tests and linkage disequilibrium. They clearly showed a genetic differentiation between strains associated to the anthropized environment and those associated to the wild, but with some inbreeding between them. The majority of strains from the anthropized environment were clustered into additional lineages of T. gondii that are common in the Caribbean. In conclusion the two environmental populations "wild" and "anthropized" were genetically well differentiated. The anthropization of the environment seems to be accompanied with a decreased diversity of T. gondii associated with a greater structure of the populations. We detected potential interpenetration and genetic exchanges between these two environmental populations. As a higher pathogenicity in human of "wild" genotypes has been described, the interpenetration of both environments leads to hybridization between strains that may be at risk for human health.

Toxoplasma gondii and Neospora caninum in wildlife: common parasites in Belgian foxes and Cervidae?

Sera from Cervidae were tested for the presence of antibodies against Neospora caninum using ELISA; and against Toxoplasma gondii using SAG1-ELISA and a commercially available agglutination test. The T. gondii seroprevalence was 52% (38/73) in roe deer (Capreolus capreolus), 0% in bred fallow deer (0/4) (Dama dama) and red deer (0/7) (Cervus elaphus). We found 2.7% of the roe deer samples and none of the bred deer samples positive for N. caninum. Brain samples from wild roe deer, red deer and red foxes (Vulpes vulpes) were tested for the presence of T. gondii and N. caninum DNA using multiplex real-time PCR. We detected T. gondii in 18.8% (57/304) of the red foxes and in 1 of the 33 deer samples. N. caninum was found in 6.6% of the red foxes and in 2 roe deer samples. Twenty-six of the T. gondii positive DNA extracts from the red fox samples were genotyped. Twenty-five were type II and only one was found to be type III.

Direct genotyping of Toxoplasma gondii in ocular fluid samples from 20 patients with ocular toxoplasmosis: predominance of type II in France.

We report the direct genotyping analysis of Toxoplasma gondii in ocular samples collected from 20 patients, as well as associated clinical and epidemiological data. This work was aimed at better understanding the impact of genotypes of Toxoplasma gondii strains on toxoplasmic retinochoroiditis. For this purpose, we studied the aqueous humor (AH) or vitreous humor (VH) of 20 patients presenting with ocular toxoplasmosis (OT) in 2 hospitals in France. Genetic characterization was obtained with microsatellite markers in a multiplex PCR assay. In contrast to the results of previous studies, we found no association between atypical Toxoplasma gondii genotypes and the occurrence of OT. Considering the local epidemiological data, our OT patients seemed to be infected more frequently by ordinary type II strains found in the environment. In conclusion, direct genotyping of Toxoplasma gondii strains from aqueous or vitreous humor showed a predominance of the type II genotype in ocular toxoplasmosis; this may be due to a high exposure rate of this genotype in humans.

Rare form of semi-invasive aspergillosis in immunocompetent patient: case report.

Chronic necrotizing or semi-invasive aspergillosis represents a disease commonly occurred in patients with mild immunodeficiency. We report a case of chronic necrotizing pulmonary aspergillosis in immunocompetent patient without underlying disease. The discovery of the disease was made accidentally, by finding a nodular opacity on a routine chest X-ray. The diagnostic was confirmed by pathological and bacteriological examination. With specific antifungal treatment, no complete eradication was obtained and the patient has a slow evolution with many relapses.

Molecular and biological characteristics of Toxoplasma gondii isolates from wildlife in France.

Toxoplasma gondii isolates have been classified into 3 genetic types. Little is known about genotypes of T. gondii isolates in wild animals in Europe. In this report, genotypes of T. gondii isolates from wildlife in France are described. Sera from wildlife were tested for antibodies to T. gondii with the modified agglutination test, and the hearts from animals with titers superior or equal to 1:6 were bioassayed individually in mice. T.gondii was isolated from 9 of 14 seropositive red foxes (Vulpes vulpes), 12 of 33 roe deer (Capreolus capreolus), 1 of 4 deer (Cervus elaphus), 1 of 7 mouflons (Ovis gmelini musimon) and 1 of 2 common mallards (Anas platyrhynchos). No isolate was obtained by bioassay in mice of 1 fallow deer (Dama dama) and of 3 European brown hares (Lepus europaeus). Genotyping of the 24 isolates using PCR-RFLP and microsatellite markers indicated that all were type II and none of these Toxoplasma isolates was virulent for mice.

Toxoplasma gondii, "new" genotypes and virulence.

Toxoplasma gondii has been described as a parasite with a low genetic diversity and a clonal population structure. The three main clonal lineages designated as type I, II or III largely predominate in Europe and North America. But strains not related to these main lineages circulate, notably, in other continents. They possess a shuffled combination of alleles that typify the three clonal types and unique polymorphisms detected by multilocus analysis. The population structure of Toxoplasma in these continents is also characterized by a higher genetic diversity associated with a lower linkage desequilibrium suggesting a role for genetic exchange. Due to their genomic diversity, it is difficult to draw global conclusions about their virulence. However, most of them are virulent in mice at isolation. Several reports also suggest a higher pathogenicity in humans and an association with ocular toxoplasmosis or severe cases of acquired toxoplasmosis in immunocompetent patients.

Atypical Toxoplasma gondii strain from a free-living jaguar (Panthera onca) in French Guiana.

Like domestic cats, wild felids are involved in the complete infective cycle of Toxoplasma gondii because they can host in their gastrointestinal tract sexually mature parasites and shed infective oocysts in their feces. We report, to our knowledge, the first isolation and molecular characterization of a T. gondii strain from the heart tissue of a free-living jaguar (Panthera onca) in French Guiana. Sequencing at six polymorphic markers indicated that the jaguar isolate had an atypical genotype, including an allele at TgM-A previously found only in isolates from South America, and an allele at GRA6, which was previously reported only in Californian sea otter isolates. These findings are consistent with the recent description of atypical T. gondii strains involved in severe toxoplasmoses in immunocompetent patients in French Guiana that seemed to be linked to a neotropical forest-based cycle involving wild cats and their prey.

Recent transcontinental sweep of Toxoplasma gondii driven by a single monomorphic chromosome.

Toxoplasma gondii is a highly prevalent protozoan parasite that infects a wide range of animals and threatens human health by contaminating food and water. A markedly limited number of clonal parasite lineages have been recognized as predominating in North American and European populations, whereas strains from South America are comparatively diverse. Here, we show that strains from North America and Europe share distinct genetic polymorphisms that are mutually exclusive from polymorphisms in strains from the south. A striking exception to this geographic segregation is a monomorphic version of one chromosome (Chr1a) that characterizes virtually all northern and many southern isolates. Using a combination of molecular phylogenetic and phenotypic analyses, we conclude that northern and southern parasite populations diverged from a common ancestor in isolation over a period of approximately 10(6) yr, and that the monomorphic Chr1a has swept each population within the past 10,000 years. Like its definitive feline hosts, T. gondii may have entered South America and diversified there after reestablishment of the Panamanian land bridge. Since then, recombination has been an infrequent but important force in generating new T. gondii genotypes. Genes unique to a monomorphic version of a single parasite chromosome may have facilitated a recent population sweep of a limited number of highly successful T. gondii lineages.

Serological survey of caprine toxoplasmosis in Ethiopia: prevalence and risk factors.

The study was conducted to determine the prevalence and risk factors of toxoplasmosis in goats in Southern and central Ethiopia between October 2005 and May 2006. A total of 641 goats sera were tested using Modified Direct Agglutination Test (MAT), of which 480 (74.8% CI: 71.3, 78.2) were found to be positive. The highest prevalence was recorded in South Omo zone (82%) while the lowest was observed in East Shewa zone (62.2%). The study revealed that goats raised in southern Ethiopia are at a greater risk of acquiring T. gondii infection (OR = 2.55, CI: 1.726, 3.776; p = 0.000) than those which are raised in central Ethiopia. The prevalence of anti T. gondii antibody was significantly higher in older goats than in kids (OR = 2.33, CI: 1.490, 3.655; p < 0.0002) and in females than in males (p < 0.0007; OR = 0.68, CI: 0.542, 0.849). No significant difference was observed among goats kept under various husbandry practices. The high prevalence of toxoplasmosis in Ethiopian goats suggests a high risk of human infections. Further epidemiological investigation, isolation and genotyping of T. gondii are planned.